Project Summary Development of a vaccine that can elicit broadly neutralizing antibodies (bnAb) could protect against HIV/AIDS. BnAbs target the membrane envelope glycoprotein or m-Env on HIV-1, however typical vaccines use soluble, truncated forms of Env as a surrogate, as m-Env has been difficult to formulate. We have been developing a m-Env vaccine strategy that presents purified m-Env in multivalent form on liposomes, which we term, m-Env liposomes (MELs). MELs have been enabled in part by development of stable, well-ordered m-Env and producer cells that generate them in high yield. Preliminary Studies show that heterologous MEL immunization elicit sporadic tier 2 cross neutralizing antibody responses. Immunogenicity of natively assembled gp41 is underexplored due to current emphases on soluble Env vaccines. With MELs, immunogenicity of m-Env gp41 can now be studied with molecular precision, and without the neoepitopes associated with soluble Env. In SA1, we will immunize rabbits with MELs initially to determine how neutralizing antibody responses are affected by creation of `glycan holes' on gp41, by boosting with multivalent epitope-specific immunogens to the MPER and fusion peptide, and by boosting sequentially with heterologous Envs. Later, human Ig locus knockin mice will be immunized with m-Envs that bind tightly to an inferred germline precursor (iGL) of a novel bnAb that shows direct binding to the membrane-proximal external region (MPER) of gp41. In SA2, using B cell sorting and next generation sequencing bioinformatics, we will determine whether m-Envs with high affinity for iGL MPER bnAb are able to expand similar B cell lineages in the hu Ig mouse repertoire. Chinese donors samples will also be sorted for single B cells specific for the MPER. In SA3, we will screen Envs from donors in part on the basis of affinity for gp41 bnAb precursors. Overall, we aim to obtain a better understanding of how to elicit bnAbs to gp41 that recognize a well guarded, but nevertheless vulnerable surface at the base of the HIV-1 Env spike.